Processes for Making [R]-Ethyl 4-Cyano-3 Hydroxybutyric Acid

ABSTRACT

The invention provides novel processes for making ethyl-4-cyano-3-hydroxybutyrate, e.g., (R)-ethyl 4-cyano-3-hydroxybutyric acid, and 4-cyano-3-hydroxybutyric acid. The invention provides protocols for making and 4-cyano-3-hydroxybutyric acid and ethyl-4-cyano-3-hydroxybutyrate by whole cell processes, cell lysate processes, “one pot processes” and “multi-pot” processes using a variety of parameters.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/461,925, filed Jun.13, 2003, which claims the benefit of priority under 35 U.S.C. §119(e)of U.S. Provisional Patent Application Ser. No. 60/389,317, filed Jun.13, 2002, and U.S. Ser. No. 60/392,944, filed Jun. 28, 2002. Theseapplications are hereby incorporated by reference into the subjectapplication in their entireties for all purposes.

TECHNICAL FIELD

This invention relates to synthetic chemistry. In alternative aspects,the invention provides novel processes for making anethyl-4-cyano-3-hydroxybutyrate, e.g.,(R)-ethyl-4-cyano-3-hydroxybutyrate, and a 4-cyano-3-hydroxybutyricacid. The invention provides protocols for making and4-cyano-3-hydroxybutyric acid and ethyl-4-cyano-3-hydroxybutyrate bywhole cell processes, cell lysate processes, “one pot processes” and“multi-pot” processes using a variety of parameters.

BACKGROUND

Atorvastatin, e.g., LIPITOR™, is used as a selective and competitiveinhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)reductase, the rate-limiting enzyme that converts3-hydroxy-3-methylglutaryl-coenzyme A to mevalonate, a precursor ofsterols such as cholesterol. The conversion of HMG-CoA to mevalonate isan early and rate-limiting step in cholesterol biosynthesis.Atorvastatin is indicated, in conjunction with dietary restriction, inthe management of hyperlipidemia, including hypercholesterolemia, mixeddyslipidemia, and homozygous familial hypercholesterolemia.

(R)-ethyl 4-cyano-3-hydroxybutyrate is a key intermediate in thesynthesis of atorvastatin.

Crosby, John A.; Parratt, Julian S.; Turner, Nicholas J., Dep. Chem.,Univ. Exeter, Exeter, UK. Tetrahedron: Asymmetry (1992), 3(12),1547-1550, reported enzymatic hydrolysis of prochiral dinitriles, wherea series of prochiral 3-hydroxyglutaronitrile derivatives wereenzymatically hydrolyzed to corresponding nitrile-carboxylic acids withenantiomeric excesses of 22% to 84%. The products were of the(S)-configuration in all cases.

Effenberger, Franz; Osswald, Steffen., Institut fur Organische Chemie,Universitat Stuttgart, Stuttgart, Germany. Synthesis (2001), (12),1866-1872, reported selective hydrolysis of aliphatic dinitriles tomonocarboxylic acids by a nitrilase from Arabidopsis thaliana expressedin E. coli. Conversion rate and selectivity of the hydrolysis ofdinitriles w-cyanocarboxylic acids depended on the chain length.

SUMMARY

The invention provides a novel process for a key intermediate toatorvastatin (e.g., LIPITOR™), or (R)-ethyl 4-cyano-3-hydroxybutyrate((R)-ethyl 4-cyano-3-hydroxybutyric acid). In one aspect, the process ofthe invention uses epichlorohydrin as starting material and yields thetarget product (R)-ethyl 4-cyano-3-hydroxybutyric acid (i.e., (R)-ethyl4-cyano-3-hydroxybutyrate). In one aspect, the processes of theinvention yield a product with a chiral purity of more than 99% ee.

The invention provides protocols for making(R)-ethyl-4-cyano-3-hydroxybutyrate by both “one pot processes” and“multi-pot” processes using a variety of parameters, as describedherein.

The invention provides methods for making 4-cyano-3-hydroxybutyric acidcomprising the following steps: (a) providing a 3-hydroxyglutaronitrile,or equivalent; (b) providing a polypeptide having a nitrilase activity;and (c) catalyzing the conversion of the 3-hydroxyglutaronitrile, orequivalent to 4-cyano-3-hydroxybutyric acid by contacting the3-hydroxyglutaronitrile, or equivalent, with the polypeptide having anitrilase activity.

The invention provides methods for making 4-cyano-3-hydroxybutyric acidcomprising the following steps (a) providing an epichlorohydrin, orequivalent; (b) providing a polypeptide having a nitrilase activity; (c)converting the epichlorohydrin to 3-hydroxyglutaronitrile; and (d)catalyzing the conversion of the 3-hydroxyglutaronitrile to4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrilewith the polypeptide having a nitrilase activity.

The invention provides methods for making ethyl-4-cyano-3-hydroxybutyricacid comprising the following steps (a) providing a3-hydroxyglutaronitrile, or equivalent; (b) providing a polypeptidehaving a nitrilase activity; (c) catalyzing the conversion of the3-hydroxyglutaronitrile, or equivalent to 4-cyano-3-hydroxybutyric acidby contacting the 3-hydroxyglutaronitrile, or equivalent, with thepolypeptide having a nitrilase activity; and (d) converting the4-cyano-3-hydroxybutyric acid to ethyl-4-cyano-3-hydroxybutyric acid.

The invention provides methods for making atorvastatin comprising thefollowing steps (a) providing a 3-hydroxyglutaronitrile, or equivalent;(b) providing a polypeptide having a nitrilase activity; (c) catalyzingthe conversion of the 3-hydroxyglutaronitrile, or equivalent to4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile,or equivalent, with the polypeptide having a nitrilase activity; (d)converting the 4-cyano-3-hydroxybutyric acid toethyl-4-cyano-3-hydroxybutyric acid; and (e) converting theethyl-4-cyano-3-hydroxybutyric acid to atorvastatin.

In one aspect, the epichlorohydrin is converted to3-hydroxyglutaronitrile by cyanide treatment.

In one aspect, the polypeptide having a nitrilase activity is anitrilase, or, a catalytic antibody. In one aspect, the polypeptidehaving a nitrilase activity is a peptidomimetic.

In one aspect, the polypeptide having a nitrilase activity is affixed toa solid support, e.g., glass, ceramic, quartz, Sepharose, gelatin,glutaraldehyde, chitosan-treated glutaraldehyde, albumin-glutaraldehyde,chitosan-Xanthan, toyopearl gel (polymer gel), alginate,alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magneticagarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEGhydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.

In one aspect, the methods further comprise isolating the4-cyano-3-hydroxybutyric acid by precipitation with calcium hydroxide.In one aspect, the methods further comprise a step of isolating the4-cyano-3-hydroxybutyric acid by distillation, column chromatography,crystallization or precipitation. In one aspect, the precipitation stepcomprises a Ca²⁺ salt precipitation, e.g., where the calcium saltcomprises CaCl₂. In one aspect, the crystallization step comprises useof a potassium salt, a sodium salt, a lithium salt, a rubidium salt, acesium salt, a monovalent salt or a divalent metal salt.

In one aspect, the methods further comprise one or more steps ofrecrystallizing the 4-cyano-3-hydroxybutyric acid. In one aspect, theiterative recrystallizing results in product with progressively higherdegrees of enantiospecificity, e.g., 95% ee, 99% ee, 99.5% ee or higher.In one aspect, the recrystallization step comprises use of a sodiumsalt, a lithium salt, a rubidium salt, a cesium salt, a monovalent saltor a divalent metal salt.

In one aspect, the methods further comprise conversion of the4-cyano-3-hydroxybutyric acid to ethyl-4-cyano-3-hydroxybutyric acid. Inone aspect, the methods further comprise conversion of the4-cyano-3-hydroxybutyric acid to ethyl-4-cyano-3-hydroxybutyric acid byesterification. In one aspect, the esterification comprises a Fisheresterification reaction, a transesterification reaction or anesterification under Mitsunobu conditions.

In one aspect, a product of a method of the invention is an(R)-enantiomer, or, an (S)-enantiomer, e.g., in one aspect the ethyl4-cyano-3-hydroxybutyric acid is an (R)-ethyl 4-cyano-3-hydroxybutyricacid or an (S)-ethyl 4-cyano-3-hydroxybutyric acid.

In one aspect, the (R)-ethyl 4-cyano-3-hydroxybutyric acid product hasan enantiomeric purity of between about 80% ee to 99.5% ee, betweenabout 90% ee to 99% ee, or 99% ee, 99.5% ee or higher.

In one aspect, the reaction is a one-pot reaction, or, at least twosteps of the reaction take place in sequential pots (a “pot” can be anycontainer or equivalent, e.g., a whole cell, such as a bacterial or amammalian cell). In one aspect, at least one step of the reaction takesplace in a whole cell. The cell can be encapsulated, e.g., in a gel. Inone aspect, at least one step of the reaction takes place in a wholecell in a fermentor. In one aspect, at least one step of the reactiontakes place in a cell lysate or a whole cell paste. In one aspect, atleast one step of the reaction takes place in a membrane reactor, acapillary, or a column. The capillary can comprise a capillary array,e.g., a GIGAMATRIX™.

In one aspect, the methods further comprise converting the4-cyano-3-hydroxybutyric acid to its potassium salt by reaction with apotassium hydroxide in water, methanol or ethanol. In one aspect, themethods further comprise crystallization of the 4-cyano-3-hydroxybutyricacid potassium salt.

In one aspect, the preparation of hydroxyglutaronitrile fromepichlorohydrin is followed by the addition of a neutralizing agent, abuffer or a diluent, and then subsequent conversion to4-cyano-3-hydroxybutyric acid with the nitrilase. The preparation ofhydroxyglutaronitrile from epichlorohydrin, the addition of aneutralizing agent, a buffer or a diluent, and the conversion to4-cyano-3-hydroxybutyric acid with the nitrilase can take place in asingle pot (container), or each step can be in a different pot(container), or, a combination thereof.

In one aspect, at least one step of the reaction takes place underconditions comprising a temperature in a range between about 4° C. andabout 80° C., a range between about 10° C. and about 40° C., or a rangebetween about 22° C. and 37° C.

In one aspect, at least one step of the reaction takes place underconditions comprising a pH in a range between about pH 4 and about pH11, or, in a range between about pH 5 and about pH 9.

In one aspect, substrate concentrations are in the range of betweenabout 5 M, or higher, to 10 mM, or lower.

In one aspect, the nitrilase used in a method of the invention is anisolated or recombinant polypeptide having a sequence at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete sequenceidentity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, or enzymatically active subsequences thereof.

In one aspect, the nitrilase comprises a sequence as set forth in SEQ IDNO:196, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210 or SEQ ID NO:238 andhaving one or more mutations selected from the group consisting of amutation at residue 55 lysine, residue 55 glycine, residue 55 glutamine,residue 60 glutamic acid, residue 111 serine, residue 190, residue 190serine, residue 190 histidine, residue 190 tyrosine, residue 190threonine, residue 191 leucine, residue 191 valine, residue 191methionine, residue 191 aspartic acid, residue 191 glycine, residue 191glutamic acid, residue 191 tyrosine, residue 191 threonine, residue 199glutamic acid, residue 199 leucine, residue 222 leucine, and anycombination thereof.

In one aspect, the nitrilase comprises a sequence as set forth in SEQ IDNO:196, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210 or SEQ ID NO:238 andhaving a mutation at residue 190 or equivalent, wherein alanine isreplaced with a hydrogen-binding amino acid or hydrogen-bindingpeptidomimetic residue.

In one aspect, the nitrilase comprises a sequence as set forth in SEQ IDNO:196, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210 or SEQ ID NO:238 andhaving a mutation at residue 190 or equivalent, wherein alanine isreplaced with a hydrophobic amino acid or hydrophobic peptidomimeticresidue.

In one aspect, the nitrilase comprises a sequence having the equivalentof one or more mutations at residue 55 lysine, glycine, or glutamine; atresidue 60 glutamic acid; at residue 111 serine, at residue 190, serine,histidine, tyrosine or threonine; at residue 191, leucine, valine,methionine, aspartic acid, glycine, glutamic acid, tyrosine orthreonine; at residue 199 glutamic acid or leucine; at residue 222leucine of SEQ ID NO:196, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210 orSEQ ID NO:238.

In one aspect, the nitrilase is encoded by a nucleic acid having asequence at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383 or 385.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 schematically illustrates an exemplary method of the inventionusing epichlorohydrin to yield the product (R)-ethyl4-cyano-3-hydroxybutyric acid.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The methods of the invention use epichlorohydrin, or equivalent, as astarting material to yield the product(R)-ethyl-4-cyano-3-hydroxybutyrate. In one aspect, the product isproduced with a chiral purity of more than 99% ee.

In one aspect of the processes of the invention, epichlorohydrin isconverted to 3-hydroxyglutaronitrile (HGN) by cyanide treatment under pHand temperature control, with high yield and purity. In one aspect ofthe processes of the invention, 3-hydroxyglutaronitrile (HGN), whetherprepared by this method or another process, whether a crude product(e.g., without isolation) or an isolated pure composition, is convertedto 4-cyano-3-hydroxybutyric acid by catalysis with a nitrilase. Anynitrilase can be used.

In one aspect, the processes of the invention produce anenantiomerically pure product, e.g., an(R)-ethyl-4-cyano-3-hydroxybutyrate. In alternative aspects of theprocess, the product is 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%or 100% enantiomerically pure, for example the processes of theinvention yielding a product having an enantiomeric excess of betweenabout 85% ee to about 99.5% ee. In one aspect, the chiral product is an(R)-enantiomer. In another aspect, the chiral product is an(S)-enantiomer.

In one aspect, the processes of the invention yield a product having anenantiomeric excess of between about 90% ee to 99% ee. It wasdemonstrated that this acid product (4-cyano-3-hydroxybutyric acid) canbe isolated via precipitation with calcium hydroxide. The precipitatecan then be converted into (R)-ethyl-4-cyano-3-hydroxybutyrate byesterification, in a one-pot reaction.

In another aspect, the acid product can be converted to its potassiumsalt by reaction with potassium hydroxide in water, methanol or ethanol.A process is established whereby crystallization of this potassium saltfrom ethanol or methanol/MTBE is shown to upgrade the enantiomericexcess of the acid to greater than 99% ee. In one aspect, the process iscarried out with and/or without the crystallization step or with andwithout a Ca²⁺ salt precipitation step.

In another aspect, the methods include the sequential preparation ofhydroxyglutaronitrile (HGN) from epichlorohydrin followed by theaddition of neutralizing agent, buffer or other diluents and thensubsequent transformation with a nitrilase or the direct addition of anitrilase to the crude reaction of the HGN production.

In another aspect, a 4-cyano-3-hydroxy butyrate ester may be made (inanother aspect, in the same pot) through a variety of methods (seebelow).

In another aspect, sequential pots are used for two or more or all orthe different steps of the reaction. In one aspect, crude preparations,or purified materials, or both, are transferred to new vessels.

In one aspect, the nitrilase reaction is applied to purify crudehydroxyglutaronitrile (HGN). In alternative aspects, purification can beby distillation, column chromatography, crystallization (e.g. K+ salt)or precipitation (e.g. CaCl₂).

In alternative aspects, various types of crystallizing salts (not onlypotassium) can be used. Other types of crystallizing salts can be usedinclude sodium, lithium, rubidium, cesium or any other monovalent ordivalent metal.

The invention provides several ways of doing esterification, includingFisher esterification (e.g., Fisher-Speier esterification, see, e.g.,Srinivas (1990) J. Chromatogr. 530:327-336), catalyzed by HCl, sulfuricacid, or other acids including acidic resins; or, esterification underMitsunobu conditions (see, e.g., Ahn (2002) J. Org. Chem. 67:1751-1753;Ahn (2002) J. Org. Chem. 67:1754-1759). The methods of the invention canbe practiced with any protocol for the preparation of esters, includingunder preparation of esters Mitsunobu conditions or any other methodknown in the art, including transesterification. In alternative aspects,the methods of the invention can be practiced with other esters ifpossible: methyl, ethyl, propyl, iso-propyl, butyl etc.

In alternative aspects, the invention provides various preparations oftert-butyl esters by treatment of the salt with tert-butyl bromide andthe preparation of esters by treatment of carboxylic acid withdehydrating agents (e.g. DCC-dicyclohexylcarbodimide) and an alcohol.

In alternative aspects, processes of the invention can be performeddirectly in a fermentor with whole cells, with whole cell paste (e.g.,wet or dry, immobilized or free), using cell lysates, e.g., crude,semi-processed, unprocessed or clarified lysate, lyophilized or not, orimmobilized or free lysate preparations.

The methods of the invention can be practiced with purified or partiallypurified nitrilase. The nitrilase can be encapsulated within a gel. Ifwhole cells are used, they can be encapsulated within a gel.

In alternative aspects, the processes of the invention can be run in abatch wise process or via pulsed or continuous feeding of substrate in anumber of reactor configurations, including a membrane reactor orcolumn.

In alternative aspects, the processes of the invention can be run usingtemperatures in the range of 2° C., 4° C., 10° C., 15° C., 22° C., 30°C., 37° C., 40° C. (e.g., in a range of between about 4° to about 40°C.), 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., to protectas high as 80° C. or more. The processes of the invention can be runusing pH in the range of about 5 to about 9, or, from about pH 4 toabout pH 11, at a pH of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 orhigher, or lower.

In alternative aspects, the processes of the invention can be run usingsubstrate concentrations as high at 5 M and as low as 10 mM, e.g., at asubstrate concentration of about 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600mM, 700 mM, 800 mM, 900 mM, 1 M, 1.5 M, 2 M, 2.5 M, 4 M, 3.5 M, 4 M, 4.5M 5 M, or higher, or lower.

In alternative aspects, the processes of the invention can be run usingvarious ionic strengths and buffer conditions. The process of theinvention can be run using no buffer under pH stat conditions or insaline unbuffered.

General Methods

In various aspects, the invention provides processes for making4-cyano-3-hydroxybutyric acid and ethyl-4-cyano-3-hydroxybutyrate. Theinvention can be practiced in conjunction with any method or protocolknown in the art, which are well described in the scientific and patentliterature.

The skilled artisan will recognize that the starting and intermediatecompounds used in the methods of the invention can be synthesized usinga variety of procedures and methodologies, which are well described inthe scientific and patent literature., e.g., Organic SynthesesCollective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY;Venuti (1989) Pharm Res. 6:867-873. The invention can be practiced inconjunction with any method or protocol known in the art, which are welldescribed in the scientific and patent literature. Enzymes of theinvention, and the enzymes used in the methods of the invention, can beproduced by any synthetic or recombinant method, or, they may beisolated from a natural source, or, a combination thereof.

The compositions used to practice the invention, including nucleic acidsand proteins used to practice the methods of the invention can bedetected, confirmed and quantified by any of a number of means wellknown to those of skill in the art. General methods for detectingreaction products of the methods of the invention, starting products andintermediates, nucleic acids and proteins used to practice theinvention, and the like, include analytic biochemical methods such asspectrophotometry, radiography, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like. The detection of nucleic acidscan be by well known methods such as Southern analysis, northernanalysis, gel electrophoresis, PCR, radiolabeling, scintillationcounting, and affinity chromatography.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and embodimentswill be apparent to those of skill in the art upon review of thisdisclosure.

Nitrilases

In one aspect of the processes of the invention, 3-hydroxyglutaronitrile(HGN) is transformed into 4-cyano-3-hydroxybutyric acid catalyzed by anitrilase. Any nitrilase can be used to practice the invention.Nitrilases are capable of converting nitriles directly to carboxylicacids. Nitrilase enzymes used to practice the methods of the inventionare found in a wide range of mesophilic micro-organisms, includingspecies of Bacillus, Norcardia, Bacteridium, Rhodococcus, Micrococcus,Brevibacterium, Alcaligenes, Acinetobacter, Corynebacterium, Fusariumand Klebsiella. Additionally, nitrilase enzymes used to practice themethods of the invention include isolated and recombinant thermophilicnitrilases, some of which can be derived from bacteria.

In one aspect, a nitrilase used in a method of the invention is anisolated or recombinant polypeptide encoded by a nucleic acid having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or completesequence identity (100% identical) to SEQ ID NO:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, or variants or subsequencesthereof.

In one aspect, a nitrilase used in a method of the invention is anisolated or recombinant polypeptide having a sequence at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete sequenceidentity (100% identical) to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344,346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372,374, 376, 378, 380, 382, 384, 386, or variants thereof, or activesubsequences thereof.

Exemplary nitrilase variants that can be used in the methods of theinvention may include, for example, the following variations of SEQ IDNO:195, 205, 207, 209, or 237, having one or more mutations: atpositions 163-165 AAA, AAG, GGT, GGC, GGA, GGG, CAA, or CAG; atpositions 178-180 GAA or GAG; at positions 331-333 TCT, TCC, TCA, TCG,AGT, or AGC; at positions 568-570 CAT, CAC, TCT, TCC, TCA, TCG, AGT,AGC, ACT, ACC, ACA, TCA, TAT, TAC, ATG or ACG; at positions 571-573 TTA,TTG, CTT, CTC, CTA, CTG, GTT, GTC, GTA, GTG, ATG, ACT, ACC, ACA, GAT,GAC, GGT, GGC, GGA, GGG, GAA, GAG, TAT, TAC, or ACG; at positions595-597 GAA, GAG, TTA, TTG, CTT, CTC, CTA, or CTG; at positions 664-666TTA, TTG, CTT, CTC, CTA, or CTG; or any combination thereof.

Exemplary nitrilase variants that can be used in the methods of theinvention may also include the following variations of SEQ ID NO:196,206, 208, 210 or 238, having one or more mutations: at residue 55lysine, glycine, or glutamine; at residue 60 glutamic acid; at residue111 serine, at residue 190, serine, histidine, tyrosine or threonine; atresidue 191, leucine, valine, methionine, aspartic acid, glycine,glutamic acid, tyrosine or threonine; at residue 199 glutamic acid orleucine; at residue 222 leucine; or any combination thereof.

Exemplary nitrilases that can be used in the methods of the inventionmay also include fragments of an enzyme, e.g., as described herein. Inone aspect, the nitrilase used in the methods of the invention can be atleast 20, 20, 40, 50, 60, 70, 80, 90 or 100 or more amino acids inlength. Exemplary nitrilases that can be used in the methods of theinvention may also include peptidomimetics having nitrilase activity.The nitrilases that can be used in the methods of the invention can becodon-optimized polypeptides or fragments thereof, wherein the codonusage is optimized for a particular organism or cell or lysate.

In one aspect of the invention, the invention uses nitrilase variantshaving improved or diminished enantioselectivity, for example, in theconversion of a 3-hydroxyglutarylnitrile (HGN) to(R)-4-cyano-3-hydroxybutyrate, than one of the exemplary polypeptidesnoted herein.

Immobilized Polypeptide Solid Supports

The polypeptides having a nitrilase activity, e.g., enzymes, catalyticantibodies and fragments thereof, can be affixed to a solid support. Inone aspect, a mixture of enzymes or active fragments thereof used forvarious steps in a process of the invention are attached to a solidsupport. In one aspect, they can be immersed into a reaction pot, alysate, a process vat, and the like. The solid support can be taken outof the pot, vat, etc. along with the enzymes affixed thereto, forrepeated use. In one aspect of the invention, the solid support is acapillary, a gel, a resin, a polymer, a ceramic, a glass, a quartz, amicroelectrode and any combination thereof. High throughput apparatuscan be adapted and used to practice the methods of the invention, see,e.g., U.S. Patent Application No. 20020001809.

For example, solid supports useful in the methods of the inventioninclude gels. Some examples of gels include Sepharose, gelatin,glutaraldehyde, chitosan-treated glutaraldehyde, albumin-glutaraldehyde,chitosan-Xanthan, toyopearl gel (polymer gel), alginate,alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magneticagarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEGhydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.

Another solid support useful in the present invention are resins orpolymers. Some examples of resins or polymers include cellulose,acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50,polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.

Another type of solid support useful in the present invention isceramic. Some examples include non-porous ceramic, porous ceramic, SiO₂,Al₂O₃. Another type of solid support useful in the present invention isglass. Some examples include non-porous glass, porous glass, aminopropylglass or any combination thereof. Another type of solid support that canbe used is a microelectrode. An example is a polyethyleneimine-coatedmagnetite. Graphitic particles can be used as a solid support. Anotherexample of a solid support is a cell, such as a red blood cell.

Methods of Immobilization

There are many methods that would be known to one of skill in the artfor immobilizing antibodies, enzymes or fragments thereof, or reagentsor catalysts, onto a solid support. Some examples of such methodsinclude, e.g., electrostatic droplet generation, electrochemical means,via adsorption, via covalent binding, via cross-linking, via a chemicalreaction or process, via encapsulation, via entrapment, via calciumalginate, or via poly (2-hydroxyethyl methacrylate). Like methods aredescribed in Methods in Enzymology, Immobilized Enzymes and Cells, PartC. 1987. Academic Press. Edited by S. P. Colowick and N, O. Kaplan.Volume 136; and Immobilization of Enzymes and Cells. 1997. Humana Press.Ed. G. F. Series: Methods in Biotechnology, Ed. J. M. Walker.

Arrays

Reagents, catalysts and polypeptides (e.g., nitrilases) and the likeused to practice the invention can be immobilized by application to anarray. Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation,San Diego, Calif.; and arrays described in, e.g., U.S. PatentApplication No. 20020080350 A1; WO 0231203 A; WO 0244336 A, provide analternative apparatus for practicing the invention.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

Isolated or recombinant antibodies are used to practice the invention,e.g., catalytic antibodies having a nitrilase activity can be used inthe processes of the invention. Alternatively, antibodies can be used toisolate, identify or quantify the nitrilases or related polypeptidesused to practice the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif.; Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.)Academic Press, New York, NY (1986); Kohler (1975) Nature 256:495;Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring HarborPublications, New York. Antibodies also can be generated in vitro, e.g.,using recombinant antibody binding site expressing phage displaylibraries, in addition to the traditional in vivo methods using animals.See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997)Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods of the invention can be practiced in whole or in part in awhole cell environment. The invention also provides for whole cellevolution, or whole cell engineering, of a cell to develop a new cellstrain having a new phenotype to be used in the methods of theinvention, e.g., a new cell line comprising one, several or all enzymesneeded to practice a method of the invention. This can be done bymodifying the genetic composition of the cell, where the geneticcomposition is modified by addition to the cell of a nucleic acid, e.g.,a coding sequence for an enzyme used in the methods of the invention.See, e.g., WO0229032; WO0196551.

The host cell for the “whole-cell process” may be any cell known to oneskilled in the art, including prokaryotic cells, eukaryotic cells, suchas bacterial cells, fungal cells, yeast cells, mammalian cells, insectcells, or plant cells.

To detect the production of an intermediate or product of the methods ofthe invention, or a new phenotype, at least one metabolic parameter of acell (or a genetically modified cell) can be monitored in the cell in a“real time” or “on-line” time frame by Metabolic Flux Analysis (MFA). Inone aspect, a plurality of cells, such as a cell culture, is monitoredin “real time” or “on-line.” In one aspect, a plurality of metabolicparameters is monitored in “real time” or “on-line.”

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.

Control of physiological state of cell cultures will become possibleafter the pathway analysis. The methods of the invention can helpdetermine how to manipulate the fermentation by determining how tochange the substrate supply, temperature, use of inducers, etc. tocontrol the physiological state of cells to move along desirabledirection. In practicing the methods of the invention, the MFA resultscan also be compared with transcriptome and proteome data to designexperiments and protocols for metabolic engineering or gene shuffling,etc. Any aspect of metabolism or growth can be monitored.

Generating and Manipulating Nucleic Acids

In one aspect, the nitrilase used to practice the methods of theinvention is an isolated or a recombinantly generated nitrilase. In oneaspect, the nitrilase used to practice the methods of the invention isencoded by a nucleic acid having a sequence at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete identity (100%identical) to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295,297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351,353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379,381, 383 and/or 385, or fragments thereof.

In one aspect, the percent identity between two given sequences (nucleicacid or polypeptide) can be calculated using an algorithm such as BLAST(Altschul (1990) J. Mol. Biol. 215:403-410). When using the BLASTalgorithm for sequences no longer than 250 nucleotides or about 80 aminoacids (“short queries”), the search parameters can be as follows: thefilter is off, the scoring matrix is PAM30, the word size is 3 or 2, theE value is 1000 or more, and the gap costs are 11, 1. For sequenceslonger than 250 nucleotides or 80 amino acid residues, the defaultsearch parameters can be used.

Nucleic acids encoding enzymes used to practice the methods of theinvention, whether RNA, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems. Nucleic acidsused to practice the methods of the invention, and to make thepolynucleotides and polypeptide of the invention, can be generated usingamplification methods, which are also well known in the art, andinclude, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS,A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y.(1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y.,ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario).

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440 3444; Frenkel (1995) Free Radic. Biol. Med. 19:373 380; Blommers(1994) Biochemistry 33:7886 7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993). Anotheruseful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACS), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACS), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1

Materials: 3-hydroxyglutaronitrile was purchased from TCI America.(R)-3-hydroxy-4-cyanobutyric acid was obtained from Gateway ChemicalTechnology (St. Louis, Mo.). All other reagents were purchased fromSigma Aldrich and utilized without further purification. Silica Gel,70-230 mesh, 60 A, purchased from Aldrich, was used for chromatographicpurifications.

I. (a) Preparation of 3-hydroxy-glutaronitrile (HGN)

KCN (1.13 mol, 61.7 g) was dissolved in water (160 ml) at 20° C. The pHof the solution was adjusted to and maintained at pH 10.0 by addition of6 M HCl acid via an automated burette and below 10° C. by cooling in anice bath. Epichlorohydrin (0.60 mol, 55.5 g) was added dropwise whilethe temperature was maintained below 10° C. The reaction was allowed toproceed at 10° C. until the pH of the solution begins to drop below 10.At this point the pH was maintained at 10.0 by the addition on 6N KOHvia pH stat and the reaction was allowed to proceed overnight withstirring at 20° C. The solution was then adjusted to pH7 by addition of6 N HCl and then continuously extracted with EtOAc (400 ml) for 48 h.The organic extract was dried over Na₂SO₄ and then concentrated invacuo. Crude 3-glutaronitrile was obtained in 85% yield as a deep redliquid (57 g, 0.52 mol). The product was purified by vacuum distillationto obtain hydroxyglutarol nitrile as a clear yellow liquid in 59% yield(0.35 mol, 39 g) ¹H NMR (D₂O): 4.40 (m, 1H), 2.89 (m, 4H). ¹³C NMR(D₂O): 117.3, 61.91, 23.69. MS calc'd for [C₅H₆N₂O]: 110.05, found:110.00 [M⁺], (ESI+).

II. a) Hydrolysis of 3-hydroxy-glutaronitrile (purified) with nitrilase

3-hydroxyglutaronitrile (1 g, 9 mmol) was suspended in 37.5 mL of N₂ (g)sparged, room temperature, 100 mM sodium phosphate buffer at pH 7. Tothis 240 mM solution of HGN was added 150 mg of cell lysate normalizedfor protein content. As assessed by PAGE, the nitrilase content wasestablished to be 20% of the total protein. Therefore effectively, 30 mgof nitrilase is used in this reaction. The reaction was agitated at 100rpm. Reaction progress was monitored by TLC (1:1 EtOAc:Hexanes,R_(f)=0.32, nitrile; R_(f)=0.0, acid) and GC. After 22 h, the reactionwas filtered through a 10,000 MW cutoff filter and then concentrated invacuo. The concentrated hydrolysis solution was acidified with 1M HCland the product was continuously extracted with MTBE (30 mL). The acidproduct (R)-3-hydroxy-4-cyanobutyric acid was isolated as a yellow oil(1.15 g, 98% yield). ¹H NMR (d₆-DMSO, 500 MHz) δ 12.32 (s, 1H), 5.52 (s,1H), 4.10 (m, 1H), 2.70 (dd, 1H, J=16.8, 4.1 Hz), 2.61 (dd, 1H, J=16.9,6.3 Hz), 2.44 (dd, 1H, J=15.4, 5.3 Hz), 2.37 (dd, 1H, J=15.6, 7.8 Hz).¹³C NMR (DMSO, 298K, 125 MHz) δ 171.9, 118.7, 63.4, 41.2, 25.2 MS calc'dfor [C₅H₇NO₃]: 129.0, found: 130.0 [M+H⁺], (ESI+). ee=95% [HPLC]. Basedon the isolated yield a productivity of 40 g g⁻¹ d⁻¹ is calculated.

b) Integrated process for preparation of HGN, hydrolysis of3-hydroxy-glutaronitrile (crude) with nitrilase, Ca(OH)₂ precipitationand esterification

KCN (1.13 mol, 61.7 g) was dissolved in water (160 ml) at 20° C. The pHof the solution was adjusted to and maintained at pH 10.0 by addition of6 M HCl acid via an automated burette and below 10° C. by cooling in anice bath. Epichlorohydrin (0.60 mol, 55.5 g) was added dropwise whilethe temperature was maintained below 10° C. The reaction was allowed toproceed at 10° C. until the pH of the solution begins to drop below 10.At this point the pH was maintained at 10.0 by the addition on 6N KOHvia pH stat and the reaction was allowed to proceed overnight withstirring at 20° C. The solution was then adjusted to pH7 by addition of6 N HCl. The final concentration of HGN in this reaction was assessed tobe 1.24 M

A portion of this crude solution of HGN (7.4 mL) was diluted with 33.6mL pH 7 100 mM phosphate. To this solution was added 100 mg of celllysate, normalized for protein content, which had been suspended in 5 mL100 mM phosphate buffer. The reaction mixture contained 9.25 mmol HGN in46 mL buffer at a final concentration of 200 mM As assessed by PAGE, thenitrilase content was established to be 20% of the total protein.Therefore effectively, 20 mg of nitrilase is used in this reaction. Thereaction was agitated at room temperature at 100 rpm. Reaction progresswas monitored by TLC (1:1 EtOAc:Hexanes, R_(f)=0.32, nitrile; R_(f)=0.0,acid) After all the HGN was consumed (22 hours), the reaction solutionwas filtered through celite.

The filtered solution was cooled in an ice-water bath and then 3 gcalcium hydroxide monohydrate was added to it. Ethanol (85 mL) was addedto the above suspension and after stirring for 1 hour the resultingwhite solid was filtered, washed with ethanol and dried in vacuoovernight. The calcium salt was isolated as a white solid (4.6 g). Thismaterial was suspended in 50 ml of 200 proof ethanol, cooled by anice-water-salt bath, and then 1 ml concentrated sulfuric acid was addeddropwise. Once acidified the solution was subjected to reflux for onehour. After cooling the solid impurities were removed by filtration andthe filtrate was concentrated in vacuo. Pure (R)-ethyl4-cyano-3-hydroxybutyrate (1.1 g, 79% yield based on epichlorohydrin,93% ee) was isolated as a pale yellow oil by Kugelrohr distillation.

(c) Crystallization of potassium 4-cyano-3-hydroxybutyrate

Method one: To a 1 g of 4-cyano-3-hydroxybutyric acid in 10 mL water,chilled on an ice bath, was added a concentrated solution of KOH (20%w/v, 1.05 equivalents). This solution was concentrated to dryness invacuo and then the residue was recrystallized from hot ethanol. Whiteneedle crystals were formed and resulted in an enantiomeric excessenhancement from 93% ee to 97% ee with up to 80% recovery achieved todate. This was performed several times on one gram scale and once at a10 g scale.

Method Two: To a 1 g of 4-cyano-3-hydroxybutyric acid in 15 mL methanolwas added a concentrated methanolic solution of KOH (20% w/v, 1.05equivalents). To this solution was added MTBE to initiatecrystallization. This procedure yielded an enantiomeric excessenhancement from 94% ee to 98% ee with 88% yield achieved to date. Asecond recrystallization afforded an enrichment from 98% ee to 99.6% ee(80% yield). In addition, in a single crystallization 95.5% ee sampleswere enriched to 99.2% (20% yield on first attempt). The crystallizationmethods have not been optimized. This was performed several times on aone gram scale.

III (a) Synthesis of (R)-Ethyl 4-cyano-3-hydroxybutyric with HClcatalyst

A 0.2 M solution of (R)-3-hydroxy-4-cyanobutyric acid (50 mg, 0.4 mmol)in anhydrous ethanol (1.94 mL) was prepared. The ethanol solution wasadded dropwise to 1.0 ml of a 50:50 (v/v) mixture of anhydrous 1 M HClethereal solution and anhydrous ethanol over molecular sieves. Thereaction was stirred overnight at room temperature under N₂. Thereaction was monitored by TLC, (1:1 EtOAc:Hexanes, R_(f)=0.45, ester;R_(f)=0.0, acid, stained with p-anisaldehyde). After 30 h, solvent wasremoved in vacuo. The crude product was taken up in Et₂O (25 mL), washedwith 5 ml saturated bicarbonate and then 5 mL brine. The organic extractwas dried over MgSO₄, filtered and then concentrated in vacuo, yieldingthe product as a clear oil. ¹H NMR (DMSO, 298K, 500 MHz) δ 5.60 (d, 1H,J=5.58 Hz), 4.12 (m, 1H), 4.07 (q, 2H, J=7.1), 2.66 (m, 2H), 2.47 (m,2H), 1.87 (t, 3H, J=7.0). ¹³C NMR (DMSO, 298K, 125 MHz) δ 170.21,118.60, 63.40, 59.98, 41.10, 25.14, 14.02. MS calc'd for [C₇H₁₁NO₃]:157.1, found 158.2.

[M+H⁺]

(b) Synthesis of (±)-Ethyl 4-cyano-3-hydroxybutyrate with H₂SO₄ catalyst

A 1 M solution of 4-cyano-3-hydroxybutyric acid in anhydrous ethanol wasprepared and cooled in an ice bath. Several drops of concentratedsulfuric acid was added. The reaction mixture was then subjected toreflux for 4 hours to overnight. After reaction, the solvent was removedin vacuo. Ethyl ester formation was found to be quantitative.

Analytical Information

All ¹H and ¹³C NMR spectra were run on Bruker model AM-500 instruments,set at room temperature, 500 MHz and 125 MHz respectively for ¹H and¹³C. GC analysis was performed on an Agilent 6890 GC with aMacherey-Nagel Lipodex A column, using a FID detector. The HPLC analysiswas performed on an Agilent 1100 HPLC with a Daicel OD column (50×0.46cm) and the DAD detector set at 210, 220, and 250 nm. Specific rotationwas determined on a Perkin Elmer Model 341 Polarimeter, set at 589 nm,Na lamp, at room temperature, with a 100 mm path length cell.Concentrations (c) for specific rotation are reported in grams per 100ml of solvent.

Analytical Methods:

Retention Times Acid Chromatography Of enantiomers Product Column MethodDetails (min) HPLC 4-cyano-3- Daicel OD 5% isopropanol, 4.5 (R); 5.4(S)hydroxy- 50 × 4.6 95% hexane butyrate mm 1 ml/min GC Methyl 4- Lipodex A95° C., 1 ml/min 51.6 (R); 52.7(S) cyano-3- 25 m hydroxy- Macherey-butyrate Nagel

Preparation of (R)-(−)-Methyl 4-cyano-3-hydroxybutyrate

(R)-4-cyano-3-hydroxybutyric acid (658 mg, 5.1 mmol) was dissolved inmethanol (20 ml) and the solution was treated with dropwise addition ofTMS-diazomethane (2 M solution in hexane, 150 mmol) at 22° C. for 1 hrto yield the methyl ester product. The mixture was concentrated invacuo, extracted with ethyl acetate (3×40 ml) and then the combinedorganic extracts were dried over MgSO₄, filtered and concentrated invacuo giving a yellow oil (549 mg, 3.84 mmol, 74% yield). ¹H NMR (DMSO,298K, 500 MHz) δ 5.60 (d, 1H, J=5.26 Hz), 4.13 (m, 1H), 3.60 (s 3H),2.71 (dd, 1H, J=16.9, 4.2 Hz,), 2.63 (dd, 1H, J=16.9, 6.7), 2.54 (dd,1H, J=14.9, 4.9), 2.44 (dd, 1H, J=15.7, 7.9) ¹³C NMR (DMSO, 298K, 125MHz) 170.7, 118.6, 63.4, 51.4, 40.9, 25.1, Exact Mass Calculated for[C₆H₉NO₃]: 143.1, found: 144.0 [M+H⁺].

Benzoyl chloride (0.068 ml, 0.75 mmol) was added to a stirred solutionof (R)-methyl 4-cyano-3-hydroxybutyrate (72 mg, 0.5 mmol) in pyridine (2ml), at room temperature. After 19 hours, an additional 0.5 equivalentof benzoyl chloride (0.023 ml, 0.25 mmol) was added. After 24 h, 1 mlwater was added to the reaction mixture that was then extracted withdiethyl ether (3×10 ml). The combined organic extracts were washed withbrine (2×10 ml), dried over MgSO₄, filtered and concentrated in vacuo.The crude product was purified by silica gel flash chromatography(eluant hexane:ethyl acetate [2:1]) yielding a yellow oil (46 mg, 0.19mmol, 37%). ¹H NMR (DMSO, 298K, 500 MHz) δ 7.96 (d, 2H, J=7.8), 7.70 (t,1H, J=7.25), 7.56 (t, 2H, J=7.8), 5.55 (m, 1H), 3.59 (s, 3H), 3.13 (m,2H), 2.90 (m, 2H). ¹³C NMR (DMSO, 298K, 125 MHz) d 169.6, 164.5, 133.8,129.3, 128.9, 128.5, 117.3, 66.0, 51.8, 37.5, 22.2 MS calc'd for[C₁₃H₁₃NO₄]: 247.25, found: 270.3 [M+Na⁺] ee=95% [HPLC]. [α]²⁰ ₅₉₈-32.4(c=0.5, CHCl₃).

Example 2

The following is an exemplary protocol for making (R)-ethyl4-cyano-3-hydroxybutyric acid by a “one pot process” and a “multi-pot”process. Other exemplary variations to the methods of the invention arealso described, including those that can be practiced in processes ofthe invention other than the exemplary “one pot” or a “multipot” processdescribed herein.

1) This method includes the sequential preparation of HGN(3-hydroxyglutaronitrile) from epichlorohydrin followed by the additionof neutralizing agent, buffer or other diluents and then subsequenttransformation with a nitrilase or the direct addition of a nitrilase tothe crude reaction of the HGN production. In addition, an ester of4-cyano-3-hydroxy butyrate may be made in the same pot through a varietyof methods (see step 5, below).

Sequential pots can also be used where the crude of purified materialsare transferred to new vessels.

2) In one aspect, the process is carried out with and/or without thecrystallization step or with and without the Ca salt precipitation step.

3) In one aspect, the nitrilase reaction is applied to purify crude HGN.Purification can be by distillation, column chromatography,crystallization (e.g. K+ salt) or precipitation (e.g. CaCl2), or anymethod known in the art.

4) Various types of crystallizing salts (not only potassium) can beused. Other types of crystallizing salts can be used include sodium,lithium, rubidium, cesium or any other monovalent or divalent metal.

5) The invention provides several ways of performing esterification,include Fisher esterification, catalyzed by HCl, sulfuric acid, or otheracids including acidic resins.

The invention also provides for preparation of tert-butyl esters bytreatment with tert-butyl ethers of the salt with tert-butyl bromide andthe preparation of esters by treatment of carboxylic acid withdehydrating agents (e.g. DCC-dicyclohexylcarbodimide) and an alcohol.The methods of the invention can be practiced with any protocol for thepreparation of esters, including Mitsunobu conditions or any othermethod known in the art, including trans-esterification.

6) The methods of the invention can be practiced with other esters ifpossible: Methyl, ethyl, propyl, iso-propyl, butyl etc.

7) Process can be performed in directly in a fermentor with cell, withwhole cell paste (e.g., wet or dry, immobilized or free), using celllysates (e.g., crude or clarified, lyophilized or not, immobilized orfree).

The methods of the invention can be practiced with purified or partiallypurified nitrilase. Nitrilase (cells etc) can be encapsulated within agel.

8) The process of the invention can be run in a batch wise process orvia pulsed or continuous feeding of substrate in a number of reactorconfigurations, including a membrane reactor or column.

9) The process of the invention can be run using temperatures in therange of 1° C., 2° C., 4° C., 10° C., 20° C., 22° C., 30° C., 37° C.,40° C., 50° C., 60° C., 70° C., 80° C., 90° C., 95° C., 99° C. (i.e.anywhere in the range of between about 1°-99° C.).

10) The process of the invention can be run using pH in the range ofabout 5 to about 9, or, from about pH 4 to about pH 11.

11) The process of the invention can be run using substrateconcentrations as high at 5 M and as low as 10 mM.

12) The process of the invention can be run using various ionicstrengths and buffer conditions. The process of the invention can be runusing no buffer under pH stat conditions or in unbuffered saline.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for making 4-cyano-3-hydroxybutyric acid comprising thefollowing steps (a) providing a 3-hydroxyglutaronitrile, or equivalent;(b) providing a polypeptide having a nitrilase activity; and (c)catalyzing the conversion of the 3-hydroxyglutaronitrile, or equivalentto 4-cyano-3-hydroxybutyric acid by contacting the3-hydroxyglutaronitrile, or equivalent, with the polypeptide having anitrilase activity.
 2. A method for making 4-cyano-3-hydroxybutyric acidcomprising the following steps (a) providing an epichlorohydrin, orequivalent; (b) providing a polypeptide having a nitrilase activity; (c)converting the epichlorohydrin to 3-hydroxyglutaronitrile; and (d)catalyzing the conversion of the 3-hydroxyglutaronitrile to4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrilewith the polypeptide having a nitrilase activity.
 3. A method for makingethyl-4-cyano-3-hydroxybutyric acid comprising the following steps (a)providing a 3-hydroxyglutaronitrile, or equivalent; (b) providing apolypeptide having a nitrilase activity; (c) catalyzing the conversionof the 3-hydroxyglutaronitrile, or equivalent to4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile,or equivalent, with the polypeptide having a nitrilase activity; and (d)converting the 4-cyano-3-hydroxybutyric acid toethyl-4-cyano-3-hydroxybutyric acid.
 4. The method of claim 2, whereinthe epichlorohydrin is converted to 3-hydroxyglutaronitrile by cyanidetreatment. 5-15. (canceled)
 16. The method of claim 1 further comprisingconversion of the 4-cyano-3-hydroxybutyric acid toethyl-4-cyano-3-hydroxybutyric acid. 17-21. (canceled)
 22. The method ofclaim 1 wherein the reaction is a one-pot reaction.
 23. The method ofclaim 1 wherein at least two steps of the reaction take place insequential pots. 24-30. (canceled)
 31. The method of claim 1 furthercomprising converting the 4-cyano-3-hydroxybutyric acid to its potassiumsalt by reaction with a potassium hydroxide in water, methanol orethanol.
 32. (canceled)
 33. The method of claim 2, wherein thepreparation of hydroxyglutaronitrile from epichlorohydrin is followed bythe addition of a neutralizing agent, a buffer or a diluent, and thensubsequent conversion to 4-cyano-3-hydroxybutyric acid with thenitrilase. 34-40. (canceled)
 41. The method of claim 1, wherein thenitrilase is an isolated or recombinant polypeptide having a sequence atleast about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete sequence identity to SEQ ID NO:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, orenzymatically active subsequences thereof.
 42. The method of claim 41,wherein the nitrilase comprises a sequence as set forth in SEQ IDNO:196, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210 or SEQ ID NO:238 andhaving one or more mutations selected from the group consisting of amutation at residue 55 lysine, residue 55 glycine, residue 55 glutamine,residue 60 glutamic acid, residue 111 serine, residue 190, residue 190serine, residue 190 histidine, residue 190 tyrosine, residue 190threonine, residue 191 leucine, residue 191 valine, residue 191methionine, residue 191 aspartic acid, residue 191 glycine, residue 191glutamic acid, residue 191 tyrosine, residue 191 threonine, residue 199glutamic acid, residue 199 leucine, residue 222 leucine, and anycombination thereof.
 43. The method of claim 41, wherein the nitrilasecomprises a sequence as set forth in SEQ ID NO:196, SEQ ID NO:206, SEQID NO:208, SEQ ID NO:210 or SEQ ID NO:238 and having a mutation atresidue 190 or equivalent, wherein alanine is replaced with ahydrogen-binding amino acid or hydrogen-binding peptidomimetic residue.44. The method of claim 41, wherein the nitrilase comprises a sequenceas set forth in SEQ ID NO:196, SEQ ID NO:206, SEQ ID NO:208, SEQ IDNO:210 or SEQ ID NO:238 and having a mutation at residue 190 orequivalent, wherein alanine is replaced with a hydrophobic amino acid orhydrophobic peptidomimetic residue.
 45. The method of claim 41, whereinthe nitrilase comprises a sequence having the equivalent of one or moremutations at residue 55 lysine, glycine, or glutamine; at residue 60glutamic acid; at residue 111 serine, at residue 190, serine, histidine,tyrosine or threonine; at residue 191, leucine, valine, methionine,aspartic acid, glycine, glutamic acid, tyrosine or threonine; at residue199 glutamic acid or leucine; at residue 222 leucine of SEQ ID NO:196,SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210 or SEQ ID NO:238.
 46. Themethod of claim 1, wherein the nitrilase is encoded by a nucleic acidhaving a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete sequence identity toSEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301,303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329,331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357,359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383 or 385.47. (canceled)